Early detection of flaviviruses using the ns1 glycoprotein

ABSTRACT

The invention concerns a method for early detection of a flavivirus-induced infection, comprising the detection of the flavivirus non-structural glycoprotein NS1 in a biological sample during the clinical phase of the infection, by an immunological method using at least two identical or different antibodies, the first antibody consisting of polyclonal or monoclonal antibodies pre-selected for their high affinity for said NS1 protein hexameric in shape.

The present invention relates to a method for the early detection offlaviviruses, in particular of the dengue virus, and to the applicationthereof.

Dengue is an acute febrile tropical disease and the virus which causesit is an arbovirus which is transmitted by mosquitoes. The vectors ofthe disease are mosquitoes of the Aedes genus, in particular Aedesaegypti, which most commonly leave their larvae in domestic andperidomestic areas. The responsible virus, isolated in 1951, has beenclassified into four different antigenic types (DEN1, DEN2, DEN3 andDEN4). It belongs to the Flaviviridae family, genus flavivirus.

More than two billion inhabitants live in endemic regions and the numberof individuals infected by the virus is thought to be more than 100million per year. Dengue is in particular responsible for 500 000hospitalizations and for several tens of thousands of deaths annually,mostly children.

After an incubation of five to eight days, the clinical signs generallybegin suddenly and consist of the appearance of undifferentiated fever(DF dengue fever) accompanied by severe headaches, lumbago, muscle andjoint pain and also shivering. From the third to the fifth day of thefebrile phase, a congestive maculopapular rash may appear for three tofour days (conventional dengue).

In its severe form, the infection may result in the appearance of ahemorrhagic syndrome (DHF or dengue hemorrhagic fever), characterized byincreased vascular permeability and deregulation of hemostasis.Although, in the majority of cases, the disease generally evolvesfavorably within a week, it may turn out to be fatal in the event ofhypovolemic shock (DSS or dengue shock syndrome). These complicationsmay be due to the presence of preexisting immunity, acquired inparticular during a primary infection with a heterologous dengue virus(different serotype). Specifically, two different types of serologicalresponse are identified in individuals infected with dengue: individualswho have never suffered a flavivirus infection and have not beenvaccinated against another flavivirus (yellow fever virus, Japaneseencephalitis virus for example) will exhibit a primary response,characterized by a slow appearance of antibodies specific for the virusresponsible for the infection; individuals who have already suffered aflavivirus infection (other dengue serotype for example) or have beenvaccinated against another flavivirus will exhibit a secondary response,characterized by the rapid appearance of antibodies.

The infectious agent is the dengue virus which belongs to theFlaviviridae family, to which the yellow fever virus and the Japaneseencephalitis virus also belong (T. P. Monath et al., (1996) Flavivirusesin B. N. Fields, D. M. Knipe, P. M. Howly et al. (eds.) “FieldsVirology” Philadelphia: Lippincott Raven Press Publishers). Theseviruses have a single-strand RNA with positive polarity which comprises11 000 nucleotides and which encodes a polyprotein of approximately 3400amino acids. It is separated into three structural proteins and sevennonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5, duringco-translational and post-translational cleavage by viral and cellularproteases. The NS1 nonstructural protein was identified for the firsttime in 1970 by P. K. Russel et al. (J. Immunol., (1970), 105, 838-845)and characterized in 1985 by G. W. Smith et al. (J. Gen Virol., (1985),66, 559-571). This glycoprotein, which is highly conserved in theflavivirus genus (T. P. Monath already mentioned), in particular in thefour dengue virus serotypes, exists in an intracellular form and in anextracellular form. The intracellular form is thought to be involved inthe early phases of replication of the virus (Hall R. A. et al., J.Virol. (1999), 73, 10272-10280; Rice C. M. et al., J. Virol., (1997),71, 291-298; Rice C. M. et al., J. Virol., (1996), 222, 159-168; Rice C.M. et al., J. Virol., (1997), 71, 9608-9617). Before being transportedto the plasma membrane, the NS1 protein undergoes dimerization. Inmammalian cells, but not in insect cells, a portion of the NS1 proteinis released into the extracellular medium, either primarily in the formof a soluble protein, or secondarily in a microparticulate form. When itis in a soluble form, the protein exists in the form of an oligomer, inparticular of a pentamer or of a hexamer (Crooks A. J. et al. J. Chrom.(1990), 502, 59-68 and J. Gen. Viral. (1994), 75, 3453-3460). At thecurrent time, the biological function of the NS1 protein is unknown.

Several studies suggest that the NS1 protein is immunodominant in naturein the protective immune response against flavivirus infections.Experiments carried out with a certain number of flaviviruses, such asthe yellow fever, dengue, Japanese encephalitis and tick-borneencephalitis viruses, have shown partial or total protection against alethal dose of homologous virus in animals vaccinated using the subunitNS1 protein or the NS1 protein produced by virus vectors, of thevaccinia or adenovirus type (Schlesinger et al., J. Virol (1986), 60,1153-1155; J. Gen. Virol., (1987), 68, 853-857; Falgout et al. J.Virol., (1990), 64, 4356-4363; Jacobs et al. J. Viral., (1992), 66,2086-2095 Hall et al. J. Gen. Viral., (1996), 77, 1287-1294; Konishi etal., Virology, (1991), 185, 401-410).

Passive immunization of mice with monoclonal anti-NS1 antibodies hasalso made it possible to obtain a certain degree of protection(Schlesinger et al., J. Immunol. (1985), 135, 2805-2809; Gould et al.,J. Gen. Virol., (1986), 67, 591-595; Henchal et al., J. Gen. Virol.,(1988), 69, 2101-2107). The role of anti-NS1 antibodies in theprotection is not entirely known. It may be that the NS1 proteins at thesurface of infected cells are recognized by complement-fixingantibodies, leading to lysis of the infected cells (Schlesinger et. al.,Virology, (1993), 192, 132-141).

No specific treatment exists and the care given to the patient isuniquely symptomatic. In the case of conventional dengue, the treatmentis based on the administration of analgesics and antipyretics. In thecase of DHF, the treatment consists of an infusion to compensate for theplasma leakage, combined with correction of hydroelectric problems andreinitiation of diuresis.

There is no commercially available vaccine against the dengue virus. Onthe other hand, protection assays with attenuated strains of the 4dengue virus serotypes have been carried out by N. Bhamarapravati et al.(Dengue and Dengue haemorrhagic fever (1997), 367-377), withunsatisfactory results. Prevention is therefore based solely oncombating the vector. This combat combines larval destruction and“adulticide” spraying.

In the absence of a vaccine, it is necessary to monitor epidemics and toprevent the above-mentioned complications; to do this, active monitoringprograms have in particular been set up by the World HealthOrganization, and essentially comprise the monitoring of cases of feverand of vector insects, and the serological and virological screening ofindividuals having a fever and suspected of being infected with thedengue virus.

The etiology of dengue is sometimes tricky to affirm when a patientexhibits a dengue-like undifferentiated febrile syndrome, the cause ofwhich may be another arbovirus, viruses which cause eruptive fevers suchas the flu, or nonviral pathogens which are agents of diseases such asleptospirosis and even malaria. Only a laboratory test can provide thediagnosis.

At the current time, several tests exist for diagnosing dengue. However,in order to obtain an interpretable result, it is necessary to combineseveral methods:

-   -   isolation of the virus, by conventional virology techniques, in        particular by infection of cell cultures or propagation in the        brain of young mice or amplification by inoculation into        mosquitoes, and examination, for example, by immunofluorescence.        These methods have the drawback of being difficult to carry out        and of depending on the sample being taken early and on good        conditions of conservation; in addition, the first results        cannot be obtained in less than a week; in order to overcome        these drawbacks, use may be made of an RT/PCR test (V. Deubel,        L'eurobiologiste (1897), volume XXXI, 37-155); however, this        means is not always reliable and cannot be used routinely in the        countries to which the dengue virus relates, for reasons of cost        and equipment;    -   serological tests; the earliest serological diagnosis consists        in searching for IgMs specific for viral antigens using the        MAC-ELISA (immunoglobulin M Antibody Capture Enzyme-Linked        ImmunoSorbent Assay) technique. Detection of these IgMs several        days after the beginning of the symptoms makes it possible to        establish a diagnosis of probability of infection with a        flavivirus. Antibodies of the IgG type appear later than        antibodies of the IgM type. In all cases, the search for        antibodies requires two samples: one at the beginning of the        clinical signs, the other 10 to 28 days later, so as to        demonstrate serological conversion via an inhibition of        hemagglutination reaction (IHA) or by ELISA.

Simple and inexpensive immunological tests have also been proposed,which can be used in the countries at risk and which use, as a specificimmunological reagent, peptides derived from the NS1 nonstructuralprotein characteristic of flaviviruses. Thus, U.S. Pat. No. 5,824,506describes a method using peptides derived from the NS1 nonstructuralprotein, which makes it possible to detect the antibodies induced by thepresence of the dengue virus; however, the peptides selected essentiallyrecognize samples obtained from convalescent individuals and alsorecognize patients infected for the second time better than thoseinfected for the first time; these disappointing results may beexplained by the fact that the peptides used are not representative ofthe antigenic characteristics of the native protein and therefore leadto poor recognition of the antibodies being sought.

In all cases, only late confirmation of an infection with a flavivirusmay be given.

A report from the Sir Albert Sakzewski Virus Research Center, RoyalChildren's Hospital, (A. Falconer, 1991) describes the search for theNS1 nonstructural glycoprotein in the serum of patients infected withthe DEN2 virus. The authors of this report have developed adouble-sandwich ELISA assay in which a rabbit serum containingpolyclonal anti-NS1 antibodies, used as capture antibodies, isimmobilized on a microtitration plate. The antigen captured is detectedusing mouse monoclonal antibodies directed against the NS1 protein,either of the dengue virus of the DEN2 type, or specific for theserological complex of dengue; the formation of the antigen/antibodycomplex is revealed using peroxidase-conjugated goat anti-mouse IgG.With this method, the authors have shown, by using the degraded orpurified dimeric NS1 protein as the standard, that the sensitivity ofdetection of the assay is approximately 4 ng/ml with the DEN2 monoclonalantibodies as the revelation probe and approximately 60 ng/ml with thegroup monoclonal antibodies.

However, this assay does not make it possible to detect the NS1 proteineither in the case of primary infections in the acute or convalescentphase, or in secondary infections in the convalescent phase in whichthere is a high titer of anti-NS1 antibodies; the authors have concludedtherefrom that the NS1 protein must be present in large amounts only incases of secondary infections, this being transiently, during theinfection.

Now, the inventors have developed a method for purifying the NS1 proteinof a flavivirus, in the hexameric form, which has allowed them to selectantibodies specific for this protein in hexameric form, and to show,surprisingly, that these antibodies are tools of choice fordemonstrating the various problems of the circulating NS1 protein in thecontext of an infection with a flavivirus, in particular in the earlyphases in which the specific antibody response is undetectable,especially during primary infections with the dengue virus.

Consequently, the inventors have given themselves the aim of providing amethod for the early detection of a flaviviral infection, whichcorresponds to practical needs better than the methods of the priorstate of the art, i.e. a method which is reliable, rapid and inexpensiveand which makes it possible to adapt the medical care in time.

Consequently, a subject of the present invention is a method for theearly detection of a flaviviral infection, characterized in that itcomprises detecting the NS1 nonstructural glycoprotein of a flavivirusin a biological sample, throughout the duration of the clinical phase ofthe infection, by an immunological method using at least two antibodies,which may be identical or different,

-   -   the first antibody or antibody for capturing the NS1        glycoprotein consisting of antibodies chosen from the group        consisting of:    -   polyclonal antibodies preselected by immunocapture on the NS1        protein of said flavivirus, in the hexameric form, and    -   mixtures of anti-NS1 monoclonal antibodies preselected for their        high affinity for the NS1 protein of said flavivirus, in the        hexameric form, said monoclonal antibodies then being purified,    -   the second antibody or revelation antibody being chosen from the        group consisting of:    -   polyclonal antibodies directed against the NS1 protein in the        hexameric form, and    -   a mixture of monoclonal antibodies directed against the NS1        protein in the hexameric form.

For the purpose of the present invention, the expression “hexameric formof the NS1 protein of a flavivirus” is intended to mean the nativeprotein obtained from the culture supernatant of mammalian cellsinfected with said flavivirus or transformed using an expression systemcomprising the gene of the NS1 protein of said flavivirus, and purifiedaccording to the method of the invention as described below. Thishexameric form of said NS1 protein, which differs from other forms suchas the monomeric form or the dimeric form of said protein, isdemonstrated using electrophoresis or chromatography techniques such asthose described in FIG. 1.

For the purpose of the present invention, the expression “polyclonal andmonoclonal antibodies directed against the NS1 protein of a flavivirus”is intended to mean antibodies obtained by immunizing a nonhuman mammal,

-   -   either with an NS1 protein in the hexameric form,    -   or with a live or inactivated flavivirus,        said polyclonal antibodies being selected for their affinity for        the NS1 protein in the hexameric form and purified in a single        step, and said monoclonal antibodies being preselected for their        high affinity for the NS1 protein in the hexameric form and then        purified by conventional techniques, in particular by ion        exchange or affinity chromatography.

For the purpose of the present invention, the expression “affinity of amonoclonal antibody for the NS1 protein in the hexameric form” isintended to mean the concentration of said protein required to saturate50% of the sites of the antibody; this is measured by the affinityconstant of said antibody, according to the protocol described inexample 5.

For the purpose of the present invention, the term “high affinity” isintended to mean an affinity for which the constant is less than 10⁻⁸ M.

Surprisingly, the use, for detecting the NS1 protein in a biologicalsample, of polyclonal antibodies selected and purified by immunocaptureon the NS1 protein in the hexameric form, or of monoclonal antibodieswhich have a high affinity for the NS1 protein in the hexameric form andwhich are purified, instead of a total hyperimmunized rabbit serum,makes it possible to significantly improve the sensitivity of the methodand to detect the NS1 protein circulating in the blood of patients, fromthe early stage of infection, both during a primary infection and asecondary infection.

The method according to the present invention has a certain number ofadvantages:

-   -   it may be carried out early: the presence of the NS1        glycoprotein is revealed during the clinical phase, before the        antibody response is detectable,    -   it is sensitive: it is possible to detect as little as less than        1 ng of protein/ml of serum, which makes it possible to detect        the circulating NS1 protein in the early phase of primary        infections,    -   it is rapid: an answer can be obtained within a day,    -   it is relatively inexpensive and can therefore be used in the        countries at risk,    -   it makes it possible to distinguish vaccinated individuals from        individuals recently infected with a flavivirus, since the NS1        protein will be absent in vaccinated individuals in which the        antibodies may still be detectable.

According to an advantageous embodiment of said method, the flaviviralinfection is an infection with the dengue virus.

According to another advantageous embodiment of said method, the firstantibody is preferably attached to a suitable solid support and thesecond antibody, is optionally conjugated to a suitable label.

According to another advantageous embodiment of said method, when thesecond antibody is not conjugated to a label, its binding to the NS1protein attached to the solid support is then detected with a thirdantibody, conjugated to a suitable label, said third antibody being aconventionally used antibody, such as for example an IgG directedagainst the second antibody and produced in particular in the goat, thepig or the donkey.

Among the labels used, mention may be made, by way of example, offluorescent labels, the biotin/streptavidin system, nonisotopic labelsor enzymes, such as for example horseradish peroxidase or alkalinephosphatase.

According to another advantageous embodiment of said method, said thirdantibody is conjugated to an enzyme.

According to another advantageous embodiment of said method,

-   -   the first antibody, or capture antibody, consists of mouse        polyclonal antibodies selected by immunocapture on the NS1        protein of the dengue virus, said protein being in the hexameric        form, and    -   the second antibody, or antibody for detecting the presence of        NS1 in the biological sample to be analyzed, consists of        polyclonal antibodies from a rabbit immunized with the NS1        protein of the dengue virus, said protein being in the hexameric        form, the attachment of said second antibody being revealed with        a third antibody, consisting of antibodies conjugated to        peroxidase and directed against the second antibody.

According to another even more advantageous embodiment of said method,the mouse polyclonal antibodies are purified by immunocapture on thehexameric NS1 protein of dengue serotype 1.

A subject of the present invention is also a kit or boxed set fordiagnosing a flaviviral infection, characterized in that it comprises:

-   -   at least one capture antibody and at least one revelation        antibody as defined above,    -   at least one positive control consisting of the NS1 protein of a        flavivirus and/or of various serotypes depending on the        flavivirus, said protein being in a hexameric form, and    -   at least one negative control consisting of a normal human        serum.

According to an advantageous embodiment of the boxed set for diagnosisaccording to the invention, said NS1 protein in the hexameric form isobtained from a culture supernatant either from infected mammalian cellsor from mammalian cells transfected with a recombined plasmid comprisingthe gene of the NS1 protein or a fragment of said gene or a fragment ofthe flaviviral genome, said fragments being capable of expressing all orpart of the NS1 protein.

According to another advantageous embodiment of the boxed set fordiagnosis according to the invention, the NS1 protein is that of thedengue virus.

According to an even more advantageous embodiment of said boxed set fordiagnosis, the plasmid is the pCIneo-NS1.FGA plasmid which was depositedwith the Collection Nationale de Cultures et de Microorganismes[National collection of cultures and microorganisms] held by theInstitut Pasteur under the number I-2220, dated Jun. 7, 1999.

A subject of the present invention is also a method for purifying theNS1 protein of a flavivirus, in the hexameric form, from a culturesupernatant either of infected mammalian cells or of mammalian cellstransfected with a recombined plasmid comprising the gene of the NS1protein of a flavivirus or a fragment of said gene or a fragment of theflaviviral genome, said fragments being capable of expressing the NS1protein in a hexameric form, characterized in that, prior to thepurification of the NS1 protein using conventional techniques such asaffinity chromatography, the soluble form of the NS1 protein isseparated from the microparticulate form of said protein, by treatmentwith a precipitating agent and then by centrifugation.

For example, the centrifugation is carried out at a speed greater thanor equal to 10 000 g.

For the purpose of the present invention, the term “precipitating agent”is intended to mean an agent which precipitates specificallymicroparticulate proteins or cellular debris, such as for examplepolyethylene glycol, said agent being used under conventional conditionswhich make it possible to separate soluble proteins and microparticulateproteins or cellular debris.

In a preferred embodiment of said purification method, the hexameric NS1protein is that of the dengue virus, in particular dengue virus serotype1.

A subject of the present invention is also an immunogenic composition,characterized in that it comprises, as the active principle, the NS1protein of a flavivirus, in the hexameric form, optionally associatedwith other proteins, in combination with at least one pharmaceuticallyacceptable vehicle.

In a preferred embodiment of the immunogenic composition according tothe present invention, the immunogenic composition comprises at leastone mixture of the NS1 proteins in the hexameric form corresponding tothe various dengue virus serotypes.

A subject of the present invention is also an immunogenic composition,characterized in that it comprises an active principle selected from thegroup consisting of:

-   -   a polynucleotide capable of expressing all or part of the NS1        protein of the dengue virus, whatever its serotype,    -   an expression system comprising at least one promoter capable of        expressing, in the host into which it is injected, a DNA        encoding the NS1 protein of the dengue virus, whatever its        serotype, said DNA expressing said protein,        in combination with at least one pharmaceutically acceptable        vehicle.

Vaccination protocols using nucleic acids are described in particular ininternational application WO 90/11092.

A subject of the present invention is the use of an NS1 protein of aflavivirus, in the hexameric form, or of a system for the expressionthereof, for preparing an immunogenic composition capable of inducingthe production of antibodies in vivo.

In a preferred method of said use, the NS1 protein is that of the denguevirus, in particular dengue virus serotype 1.

A subject of the present invention is also the use of at least onemonoclonal anti-NS1 antibody having a high affinity for the NS1 proteinin the hexameric form, said monoclonal antibodies then being purified,and modified, for manufacturing a medicinal product capable of inducingpassive immunization.

Advantageously, the modifications to the antibodies are, in particular,the selection of Fab fragments or the humanization of the antibodies.

A subject of the present invention is also the use of the NS1 protein inthe hexameric form, for selecting in vitro antibodies specific for saidNS1 protein, able to diagnose an infection with a flavivirus, at anearly stage.

In an advantageous embodiment of said use, the antibodies are polyclonalantibodies.

In another advantageous embodiment of said use, the antibodies aremonoclonal antibodies.

In another advantageous embodiment of said use, the protein is the NS1protein of the dengue virus, in particular dengue virus serotype 1.

The anti-NS1 monoclonal antibodies are advantageously obtained by fusingspleen cells from a mouse immunized with the NS1 protein in thehexameric form, with suitable myeloma cells.

A subject of the present invention is also a method for expressing apolynucleotide encoding the NS1 protein of a dengue virus, characterizedin that it comprises the expression of a polynucleotide as defined inthe sequence SEQ ID No. 1, associated with a promoter for saidpolynucleotide, in suitable eukaryotic cells.

Other characteristics and advantages of the invention appear in theremainder of the description and the examples illustrated by the figuresin which:

FIG. 1 represents the purified hexameric extracellular NS1 proteinobtained after exclusion chromatography. (a) After exclusionchromatography, the protein is concentrated to 0.5 mg/ml byultrafiltration and treated with dimethyl suberimidate (DMS) at 0, 0.5,5 and 50 mM. The products obtained are placed in a nonreducing Laemmlibuffer, separated on a 4 to 20% gradient acrylamide gel and stained withCoomassie blue. A sample treated with 50 mM DMS is heated for 3 min at95° C. before electrophoresis in order to dissociate the noncovalentoligomers. (b) The purified NS1 protein is treated overnight at 37° C.with 0.5% or 1% of n-octylglucoside (nOG) and, optionally, treated with25 mM of DMS for 1 hour. The proteins are separated without heatdenaturation on a 4 to 20% gradient acrylamide gel and detected viaimmunoblotting with a monoclonal anti-NS1 antibody from the literatureor as defined above.

FIG. 2 represents the sequence of the NS1 protein of dengue virusserotype 1, obtained with clone 4C of example 2 below, and also thecorresponding coding sequence.

FIG. 3 illustrates the results obtained by assaying the circulating NS1protein using the method of detection by capture-ELISA in patientsinfected beforehand with a dengue virus, whose sera were taken duringthe acute and convalescent phases, and also the comparison with theresults obtained using the techniques of the prior art, IHA (inhibitionof hemagglutination of dengue virus serotypes 1, 2, 3 or 4) and MACELISA (immunoglobulin M Antibody Capture Enzyme-Linked ImmunoSorbentAssay); D1 corresponds to dengue serotype 1; D2 corresponds to dengueserotype D3 corresponds to dengue serotype 3 and D4 corresponds todengue serotype 4; ID=patient's identity; 1 corresponds to the firstsample in the acute phase of the disease, 2 corresponds to the secondsample in the convalescent phase (taken 2 to 4 weeks after the first);in the capture-ELISA assay, the values are expressed as optical densityobtained for the same serum diluted 10, 30 or 90 times.

FIG. 4 illustrates the detection of the NS1 protein using thecapture-ELISA assay on sera from patients infected with dengue virusserotype 1 from French Guiana. The numbers indicated represent thenumber of patients divided up per category (positivity or negativity bycapture-ELISA and positivity or negativity for IgM).

FIG. 5 illustrates the results obtained for 4 patients from FrenchGuiana infected with dengue virus 1, from whom samples were taken dailyduring the clinical phase of the disease from D1 to D5. Each graphcorresponds to a patient with, for each day on which a sample was taken,both the results of detection of the NS1 protein with the capture-ELISAassay developed, the results of RT-PCR and the results obtained usingthe MAC-ELISA technique. The O.D. values reported were corrected foronce the value of the background noise. The positivity thresholds areindicated by the broken lines.

FIG. 6 indicates the characteristics of the anti-NS1 monoclonalantibodies F22 and G18.

FIG. 7 illustrates the detection of the NS1 protein with thecapture-ELISA assay using the monoclonal approach in comparison with thecapture-ELISA assay using the polyclonal approach as described inexample 3. The results obtained are reported in the form of opticaldensity values measured for each dilution of serum analyzed (10th, 30thor 90th) and less the mean value of the negative controls.

FIG. 8 illustrates the demonstration of the NS1 protein in the sera frompatients infected with the yellow fever virus, using an capture-ELISAassay specific for yellow fever. For each serum tested, the opticaldensity value measured using the assay developed, less the mean value ofthe negative controls, the optical density value measured using theMAC-ELISA assay specific for yellow fever IgMs and the results of theviral isolation, when they are available, are reported.

EXAMPLE 1 Purification of the NS1 Protein of Dengue Virus Serotype 1 1.Materials and Methods

The protein is produced on Vero cells infected with dengue virusserotype 1, strain FGA/89 (P. Després et al., Virol, (1993), 196,209-219), under the conditions adapted for the method described by A. K.I. Falconar et al., (J. Virol. Meth, (1990), 30, 323-332).

The culture medium is harvested 5 days after infection and centrifugedat 1500 g to remove the cellular debris. The centrifugation supernatantis brought to 20 mM Tris-HCl, 1 mM sodium azide, and concentrated 6times by cold ultrafiltration; and the viral particles are removed byprecipitation for 2 h at 4° C., with a concentration of 7.5% ofpolyethylene glycol (PEG), followed by centrifugation for 30 minutes at10 000 g. The clarified supernatant, containing 7.5% PEG is treated with0.05% Tween 20 and 1 mg/ml of aprotinin, and then passed over animmunoaffinity column to which is attached an anti-NS1 monoclonalantibody. The protein is eluted according to the diethylamine technique,as described in Falconar et al., (mentioned above), and concentrated byultrafiltration, and the elution solution is exchanged with a 10 mMTris-HCl buffer, pH 7.5, containing 1 mM of sodium azide.

2. Results

The results are illustrated in FIG. 1.

FIG. 1 a shows that the NS1 protein is indeed in the hexameric form. Theproportion of hexameric form increases with an increase in concentrationof DMS (FIG. 1 a).

The extracellular NS1 protein in the hexameric form may be transformedinto dimeric subunits in the presence of the nonionic detergentn-octylglucoside (nOG) (FIG. 1 b). After incubation overnight at 37° C.in the presence or absence of n-octylglucoside (nOG) and treatment with25 mm DMS, it is observed that, in the absence of nOG, bands are presentwhich correspond to the dimer, to the tetramer and to the hexamer, andthat, in the presence of nOG, there is partial or complete dissociationof the hexamer depending on the concentration of nOG (FIG. 1 b).

EXAMPLE 2 Expression of the NS1 Protein of Dengue Virus Serotype 1 byVero Cells 1. Materials and Methods

The pCIneo-NS1.FGA plasmid (deposited with the Collection Nationale deCultures et de Microorganismes [National collection of cultures andmicroorganisms] (CNCM) of the Institut Pasteur under the No. I-2220,dated Jun. 7, 1999) containing the gene of the NS1 protein comprisingthe gene encoding its signal peptide, preceded by a translationinitiation codon and followed by a translation termination codon, isintroduced into the competent bacterium Escherichia coli (epicurian SUREfrom Stratagene). This plasmid is amplified in bacterial culture andpurified according to the conventional technique for preparing plasmidDNA. The purified DNA is used to sequence various clones (the sequenceof clone 4C is illustrated in FIG. 2) and to transfect Vero cells usingeither a suitable mixture with cationic liposcimes, such as DOTAP(Boehringer Mannheim), or with a nonliposomal agent, such as FuGENE(Boehringer Mannheim). The FuGENE and the DNA are pre-incubated inmedium without serum for 15 min, and then the mixture is brought intocontact with a layer of Vero cells for 24 h. The cells are then rinsedwith PBS (phosphate buffered saline), fixed for 20 min at roomtemperature with a solution of PBS containing 3% of paraformaldehyde andpermeabilized for 5 min with PBS containing 0.5% Triton X-100. Thepresence of NS1 antigen is then revealed using specific antibodies,which are recognized by a fluoroscein-labeled conjugated antibody.

2. Results

It is thus possible to demonstrate a strong fluorescent signal, specificfor the NS1 viral antigen, in approximately 20% of the transfectedcells.

The expression of NS1 is thus demonstrated and stable lines may beestablished in the presence of neomycin, which is a selection marker forthe transfected cells.

FIG. 2 illustrates the sequence of the NS1 protein of dengue virusserotype 1 thus obtained, and also the corresponding coding sequence.

EXAMPLE 3 Implementation of the Capture-ELISA Technique According to theInvention in the Context of an Infection with Dengue Virus Serotype 1and Comparison with the Methods of the Prior State of the Art 1.Principle of the Capture-ELISA Technique

The NS1 viral antigen is captured using monospecific mouse polyclonalantibodies purified beforehand by immunocapture on the purifiedhexameric NS1 protein, of dengue serotype 1.

The presence of NS1 is revealed using antibodies from rabbits immunizedwith the purified hexameric NS1 protein, themselves recognized byantibodies conjugated to horseradish peroxidase.

2. Materials and Methods

a—Purification Of Mouse Polyclonal Antibodies directed against the NS1proteina₁—Attachment of the Ns1 Protein to a Membrane

The purified NS1 protein is attached by adsorption to an amphotericnylon membrane (Nytran, Schleicher & Schuell). The surface of themembrane is then saturated with bovine albumin present at a concentrateof 3% in a phosphate buffered saline solution (PBS; 10 mM phosphate; pH7.2; 150 mM NaCl). After 2 rinses in PBS, the membrane is treated withPBS containing 0.25% of glutaraldehyde for 15 min at room temperature.After 3 rinses in PBS, the membrane is neutralized with a 100 mM glycinebuffer containing 3% bovine albumin, rinsed twice in PBS and then storedat 4° C. in PBS with 1 mM sodium azide.

a₂—Purification of The Mouse Monospecific Polyclonal Antibodies (CaptureAntibodies)

-   -   production of polyclonal ascites: the brains of young Swiss mice        infected with dengue virus and moribund are ground in 9 ml of        PBS buffer. The product is centrifuged for 10 min at 10 000 g at        4° C.

The viral suspension is injected into Swiss mice, according to thefollowing calendar of events:

-   -   D0: 0.5 ml of antigen subcutaneously into the thigh,    -   D3: 0.4 ml of antigen and 0.1 ml of complete Freund's adjuvant        intraperitoneally,    -   D25: 0.5 ml of antigen intraperitoneally,    -   D26: 0.5 ml of TG180 mouse ascites, and    -   D28: 0.5 ml of antigen intraperitoneally.        The ascites are harvested on D42.

After having collected the ascites, the coagulum is allowed to form for1 hour at room temperature and then centrifugation is carried out for atleast 30 min. at 1500 g. The supernatant is left to stand overnight at4° C. The pH of the supernatant is adjusted to 4.8 with 2M acetic acidand the supernatant is then centrifuged again under the same conditions.The pH of the supernatant is then brought to 7.0-7.2 by adding a 2Nsodium hydroxide solution. The supernatant may be stored at −20° C.

Purification of the Mouse Antibodies Specific for Dengue Virus Serotype1:

The membrane is incubated for one hour at room temperature in a mixtureof polyclonal ascites directed against the 4 dengue virus serotypesprepared as described above.

After rinsing the membrane 3 times in PBS, the antibodies attached tothe NS1 protein are eluted with a diethylamine solution, pH 11.4(Dubelco medium modified with Iscove (Gibco) containing 100 mMdiethylamine). The antibodies are concentrated by ultrafiltration andreturned to a PBS buffer containing 1 mM sodium azide.

b—Preparation of Rabbit Polyclonal Antibodies Directed Against the NS1Protein (Revelation Antibodies):

The rabbits were immunized with 3 or 4 successive injections of 30 μg ofhexameric NS1 protein purified according to the method of example 1,given on D0, D7, D21 and, optionally, on D49, and followed by bleedingout on D83. The serum is depleted of nonspecific signal by incubationwith Sepharose beads bearing a monoclonal antibody described in theliterature or prepared as described above.

c—Capture-ELISA Methodc₁—Standard Curve

For each capture-ELISA plate intended for testing human sera, a standardrange is prepared from a solution of NS1 protein purified according tothe method described in point 1, the initial concentration of which is0.5 μg/ml, and which is diluted in 3-fold serial dilutions.

c₂—Detection of the Circulating Ns1 Protein During the Acute Phase:

The purified mouse polyclonal antibodies obtained according to themethod described above (capture antibodies) are attached to a plate,diluted in a PBS solution and left to incubate overnight at 4° C. After3 rinses for 5 minutes with a solution of PBS/0.05% Tween, the plate issaturated with a mixture of PBS, 0.05% Tween and 3% milk for 30 minutesat room temperature. After 3 rinses with a solution of PBS/0.05% Tween,the sera to be tested, diluted or undiluted, are deposited and left toreact for one hour, still at room temperature. The 1/10th, 1/30th and1/90th dilutions are prepared in a solution of PBS/0.05% Tween. After 3rinses, the second antibody specific for NS1 (revelation antibodyobtained in point 3 above) is added, after having been diluted in amixture of PBS/0.05% Tween and of 3% milk, and left to incubate for 45minutes at 37° C. After 3 rinses, the anti-IgG antibodies are directedagainst the second antibody and labeled with peroxidase, said antibodybeing prepared under conventional conditions known to those skilled inthe art, is added and the incubation is carried out for 45 minutes at37° C. After 3 rinses, revelation is carried out for 10 minutes with asolution of TMB (3,3′, 5,5′-tetramethylbenzidine, Kierkegaard & PerryLab). The colorimetric reaction is stopped with sulfuric acid.

3. Results

They are illustrated in FIG. 3.

The capture-ELISA technique according to the invention makes it possibleto detect the presence of NS1 protein in the acute phase of the disease,this detection being independent of whether the patients have a primaryor secondary infection.

The results confirm that the presence of the NS1 protein is transient,since this protein is not detected in the samples taken in theconvalescent phase (FIG. 3).

93% of the samples taken in the acute phase of the disease prove to benegative using the MAC ELISA assay, whereas 100% of the samples taken inthe convalescent phase prove to be positive in this same assay (FIG. 3).

Similarly, the inhibition of hemagglutination assay (IHA) does not makeit possible to detect infection with dengue virus serotype 1 in 80% ofcases in the acute phase of the disease, but this test proves to bepositive in 100% of the samples taken in the convalescent phase (FIG.3). According to the WHO criteria, an IHA level of less than 1280 in theserum taken in the convalescent phase allows diagnosis of a primarydengue infection and a level of greater than 1280 allows diagnosis of asecondary dengue infection.

Half of the positive sera in this study therefore correspond to cases ofprimary dengue and the other half to cases of secondary dengue. Thecapture-ELISA technique according to the invention thus makes itpossible to detect the NS1 protein in cases of primary and secondarydengue.

EXAMPLE 4 Determination of the Detection Window 1. Materials and Methods

a—Study Carried Out on a Population of Patients from French GuianaInfected with Dengue Virus 1

The samples are taken from patients infected with dengue virus serotype1, between D0, marking the appearance of the clinical signs (initially,a nondifferentiated lever) and D66 corresponding to the end of theconvalescent phase.

The presence of circulating NS1 is sought in the sera of these patients,according to the capture-ELISA method described in example 3, and theresult obtained is compared with the positivity for specific IgMsmeasured by MAC-ELISA, when the data are available.

b—Daily Monitoring of 4 Patients Infected with Dengue Virus 1

Samples were taken daily from 4 patients during the clinical phase fromD1 to D5. An RT-PCR reaction to reveal the viral RNA, a MAC-ELISA assayto detect IgMs specific for dengue virus and a search for the dengue NS1antigen according to the capture-ELISA method described above werecarried out on each blood sample.

2. Results

a—Determination of the Detection Window

The results are given in FIG. 4.

Between D1 and D6, the possibility of detecting the circulating NS1protein oscillates between 64% (on D2) and 100% (on D5) of the infectedpatients. Beyond D10, the circulating NS1 protein is no longer detected,whereas the antibody response becomes predominant.

Detection of the circulating NS1 protein does not appear to be dependenton the presence of total IgMs (specific for the viral antigens) whichappear, in certain cases, on D3 and culminate from D5, nor even, forcertain patients, on the presence of total IgGs which may appear fromD2. On the other hand, the absence of detection of the NS1 antigen inclinical phase sera may be explained by the presence of IgGsspecifically directed against NS1.

Thus, the detection window for the NS1 antigen in the serum, using thecapture-ELISA technique according to the present invention, ispreferably between D1 and D6 after appearance of the clinical signs.

b—Daily Monitoring of 4 Patients Infected with Dengue Virus

The results are given in FIG. 5.

In the 4 patients studied, the NS1 protein is detected continuously upto D5, this being regardless of the day on which the sample was takenrelative to the start of symptoms. For certain patients, the detectionwindow for the protein is wider than the period of viremia, detected byRT-PCR.

EXAMPLE 5 Implementation of the Capture-ELISA Technique with MonoclonalTools in the Context of an Infection with Dengue Virus Serotype 1 andComparison with the Capture-ELISA Technique Described Above 1. Materialsand Methods

a—Production and Characterization of Mouse Monoclonal AntibodiesDirected Against the NS1 Protein of Dengue Virus Serotype 1a₁—Production of Mouse Monoclonal Antibodies Directed Against the NS1Protein

Female Balb/C mice were immunized with 7 injections of 10 μg ofhexameric NS1 protein of dengue virus serotype 1, purified according tothe method of example 1. The first injection in complete Freund'sadjuvant and the subsequent five injections in incomplete Freund'sadjuvant are given subcutaneously 15 days apart. The final injection, inincomplete Freund's adjuvant, given three days before the animal issacrificed, is given intraperitoneally.

The cells from the spleen of the immunized mice are fused with the murinmyeloma and cultured until clones appear, according to standardprotocol.

a₂—Identification of Hybridomas Secreting Anti-NS1 Antibodies

Antibodies specific for the NS1 protein were detected either using aconventional ELISA technique or using a capture-ELISA technique.

Conventional ELISA Technique

The hexameric NS1 protein purified according to the method of example 1is attached to a plate by adsorption, at the concentration of 1 μg/ml ina PBS solution overnight at 4° C. After 3 washes with a solution ofPBS/0.1% Tween (PT), the protein is incubated with the supernatants ofthe various hybridomas diluted two-fold with a solution of PT containing0.5% gelatin (PTG), for 1 h at 37° C. After 3 washes with PT, theperoxidase-labeled anti-mouse IgG antibody diluted in PTG is added andincubated for 1 h at 37° C. After 3 washes, revelation is carried outwith a solution of hydrogen peroxide in the presence oforthophenylenediamine.

Capture-ELISA Technique

The technique used is described in example 3 (cf. detection of thecirculating NS1 protein in the acute phase), but replacing the dilutionsof sera to be tested with a 1/10th dilution, in the PTG solution, ofculture supernatant on uninfected Vero cells or Vero cells infected for5 days with dengue virus serotype 1, and precipitated with 7% ofpolyethylene glycol (cf. example 1: purification of the NS1 protein ofdengue virus serotype 1). The reactivity of the supernatants from thevarious hybridomas with respect to the culture supernatant fromuninfected Vero cells is used as a control for nonspecific signal.

a₃—Reactivity of the Anti-NS1 Monoclonal Antibodies, by IndirectImmunofluorescence, on Vero Cells Infected with one of the 4 DengueVirus Serotypes

The Vero cells are infected for 40 h with one of the 4 dengue virusserotypes:

-   -   serotype 1: strain FGA/89    -   serotype 2: strain NG    -   serotype 3: strain H87    -   serotype 4: strain H241

After 1 wash with a solution of PBS, the cells are fixed with a solutionof 3% paraformaldehyde in PBS for 30 minutes at laboratory temperature.The cells rinsed in PBS are then permeabilized with a solution of 0.5%Triton X-100 in PBS for 10 minutes. After rinsing in PBS, the cells areincubated for 1 h with the supernatants from the various hybridomaswhich have reacted positively by ELISA. After 3 washes with PBS, thefluorescene-labeled anti-mouse IgG antibody is added and incubated for 1h. After 3 washes in PBS, the slides are covered with a coverslip andobserved under a fluorescent microscope.

a₄—Preparation of the Mouse Monoclonal Ascites

The monoclonal ascites are produced in Balb/C mice. The mice are givenan intraperitoneal injection of 0.5 ml of pristane(2,6,10,14-tetramethylpentadecane, Sigma) one week before theintraperitoneal injection of the hybridoma clone secreting themonoclonal antibody. The ascites are removed as they form, centrifugedat 1500 rpm for 20 minutes and stored at −20° C.

a₅—Determination of the Isotype of the Anti-NS1 Monoclonal Antibodies

The isotype of the anti-NS1 antibody is determined by ELISA usingantibodies directed against the various murine immunoglobulinsubclasses: IgG1, IgG2a, IgG2b and IgG3. The light chain of theimmunoglobulin is determined according to an identical methodology.

a₆—Determination of the Affinity Constant of the Anti-NS1 MonoclonalAntibodies (B Friguet et al., J. Immunol, (1985), 77, 305-319)

The affinity of an antibody corresponds to the concentration of antigenrequired to saturate 50% of the sites of the antibodies. An incubationis carried out in liquid medium between the antibody at constantconcentration and the antigen at decreasing concentration overnight at4° C. in order to reach the equilibrium of the reaction. Theconcentration of free antibodies, after equilibrium, is determined usingan ELISA assay: the mixture is deposited onto a plate preincubated withthe antigen. After incubation for 20 minutes at 4° C. (to avoid a shiftof the equilibrium), the ELISA is revealed with aβ-galactosidase-coupled anti-mouse IgG, followed by the enzymaticreaction. The dissociation constant KD is then determined.

a₇—Competition Reaction for the Various Anti-NS1 Monoclonal Antibodies

This reaction makes it possible to determine the specificity of themonoclonal antibodies with respect to the same epitope or to differentepitopes. Epitope determination brings into play the reactivity for anantigen, of an unlabeled monoclonal antibody and of a second-monoclonalantibody, coupled to biotin.

The first monoclonal antibody, unlabeled, is placed at saturatingconcentration (determined beforehand by ELISA) on a plate to which theantigen has been attached beforehand, and incubated for 2 h at 37° C.After 4 washes in PT solution at 4° C., the second monoclonal antibody,coupled to biotin, is added and incubated for 20 minutes at 4° C. After4 washes in PT solution at 4° C., the solution of peroxidase-labeledstreptavidin conjugate is added and incubated for 1 h at 37° C. After0.4 washes in PT solution, the complex is revealed with a solution ofhydrogen peroxide in the presence of orthophenylenediamine. If a signalis obtained after reading on a spectrophotometer, this indicates thatthe epitopes recognized by the 2 antibodies are different. If theopposite is true, the 2 monoclonal antibodies are directed against thesame epitope of the antigen.

b—Purification of the Monoclonal Antibodies G18 and F22

The antibodies G18 and F22 are purified by immunoaffinity as describedin Example 3.

c—Detection of the Circulating NS1 Protein with a Capture-ELISA AssayUsing the Monoclonal Antibodies

The purified monoclonal antibodies G18 and F22 are mixed in a solutionof PBS at a given dilution and incubated overnight at 4° C. Thesubsequent steps of this ELISA assay are similar to those of theprevious example.

d—Comparison of the Capture-ELISA Assay Using the Monoclonal Approachwith that Using the Polyclonal Approach

A panel of serum from French Guiana was tested on the same day with thecapture-ELISA assay using the monoclonal approach and then using thepolyclonal approach. The sera are tested at various dilutions: 10th,30th and 90th.

2. Results

a—Characteristics of the Monoclonal Antibodies

The results are given in FIG. 6.

The antibodies G18 and F22 were selected for their ability to bind, withhigh affinity, to different epitopes of the NS1 protein. The antibodyF22 is specific for dengue virus serotype 1, and G18 is specific fordengue virus serotypes 1 and 3.

b—Use of the Monoclonal Antibodies for NS1 Antigen Capture

The results are given in FIG. 7.

The monoclonal antibodies selected not only reproduce the resultsobtained with the polyclonal approach, but they exhibit more markedreactivities than the polyclonal antibodies. The monoclonal tooldeveloped therefore appears to be particularly suitable for thediagnostic use which must be made of it.

EXAMPLE 6 Implementation of the Capture-ELISA Technique According to theInvention in the Context of an Infection with Another Dengue VirusSerotype or Another Flavivirus 1. Materials and Methods

a—Preparation of Culture Supernatants

The Vero cells are infected either with dengue virus 2 or with theJapanese encephalitis virus or the yellow fever virus. The culturesupernatants are then prepared according to the method described inexample 1.

b—Purification of the Monoclonal Antibodies Directed Against the NS1Protein of the Yellow Fever Virus and of the Japanese Encephalitis Virus

The monoclonal ascites of the antibodies 8G4, 1A5 and 2D10 (J. J.Schlesinger et al., Virology, (1983), 125, 8-17) directed against theNS1 protein of the yellow fever virus, and of the antibodies 171-2-2 and70-14-20 directed against the NS1 protein of the Japanese encephalitisvirus, are purified on protein A Sepharose CL-4B beads (PharmaciaBiotech). These monoclonal ascites are incubated overnight at 4° C. onthe protein A beads. After the beads have been rinsed 3 times inPBS/0.05% Tween, the antibodies attached to the protein A beads areeluted with a solution of glycine buffer, pH=3. They are thenconcentrated by ultrafiltration and returned to a PBS buffer containing1 mM sodium azide.

c—Detection of the NS1 Protein in the Dengue Virus 2Culture Supernatantsc₁—Antibodies Used

The capture step is carried out with a mixture of ascites of themonoclonal antibodies 3D1.4 and 1A12 (A. K. I. Falconar et al., Arch.Virology, (1994), 137, 315-326). The protein is then recognized with amixture of two rabbit antibodies: the serum obtained after immunizationwith the purified protein described in example 3 and a rabbit serumobtained after immunization with the viruses of the four dengueserotypes.

c₂—Capture-ELISA Method

The technique used is the same as that described in example 3.

d—Detection of the NS1 Protein in the Japanese Encephalitis VirusCulture Supernatantsd₁—Antibodies Used

The purified monoclonal antibodies 171-2-2 and 70-14-20 are used for thecapture step. The protein is then recognized with a mixture of two serafrom rabbits which have been immunized beforehand with recombinedproteins of the NS1 protein of Japanese encephalitis.

d₂—Capture-ELISA Method

The technique used is the same as that described in example 3.

e—Detection of the NS1 Protein in the Yellow Fever Virus CultureSupernatants and the Sera from Patients Infected with this Viruse₁—Antibodies Used

The purified monoclonal antibodies 8G4, 1A5 and 2D10 are mixed, at agiven dilution, in a solution of PBS and used as capture antibodies. Thesecond antibody specific for yellow fever NS1 used originates from aserum of a rabbit immunized beforehand against the NS1 protein of theyellow fever 17D virus (J. J. Schlesinger et al., J. immunol. (1985),135, 2805-2809).

e₂—Capture-ELISA Method

The technique used is the same as that described in example 3.

2. Results

Secretion of the NS1 protein has previously been reported in in vitrocell cultures infected with various flaviviruses, the DEN2 virus(Winkler et al., Virology (1988), 162, 187-196, Pryor et al., Virology(1993) 194, 769-780), the tick-borne encephalitis virus (Lee et al., J.Gen. Virol. (1989), 70, 335-343, Crooks et al., J. Chrom. (1990), 502,59-68, Crooks et al., J. Gen. Virol. (1994), 75, 3453-3460), theJapanese encephalitis virus (Mason, Virology (1989), 169, 354-364, Fanet. al., Virology (1990), 177, 470-476), the Murray valley encephalitisvirus (Hall et al., J. Virol. Meth. (1991), 32, 11-20) and the yellowfever virus (Post et al., Vir. Res. (1990), 18, 291-302). As theseresults were obtained using different ELISA techniques, we sought todemonstrate the protein, using the capture-ELISA technique of thepresent invention, in supernatants of infected mammalian cells.

The NS1 protein is detectable in the culture supernatants of the Verocells infected either with the DEN2 virus, with the Japaneseencephalitis virus or with the yellow fever virus.

It was also possible to demonstrate the protein, using this technique,in sera from patients infected with the yellow fever virus, asdemonstrated by the results given in FIG. 8. Among the 18 seragenerously provided by Ch. Mathiot (Institut Pasteur of Dakar), 7 arepositive by NS1 antigenemia, and, as for the DEN1 virus, detection ofthe circulating NS1 protein appears to be indifferent to the presence ofIgMs specific for yellow fever.

The capture-ELISA technique according to the present invention makes itpossible to detect the NS1 protein in the culture supernatants of cellsinfected with various flaviviruses and in the sera from patientsinfected with the yellow fever virus. Because of this, it may have adiagnostic application for detecting an infection with a flavivirusother than the DEN1 virus.

1-20. (canceled)
 21. A method for purifying an NS1 protein of aflavivirus, wherein the NS1 protein is in hexameric form, from a culturesupernatant either of infected mammalian cells or of mammalian cellstransfected with a recombined plasmid, comprising: (A) expressing theNS1 protein or a fragment of the NS1 protein from an NS1 gene or afragment of the flaviviral genome, wherein the fragments are capable ofexpressing the NS1 protein prior to the purification of the NS1 protein;(B) treating the NS1 protein with a precipitating agent; (C)centrifuging the treated NS1 protein; and, (D) separating a soluble formof the NS1 protein from a microparticulate form of NS1 protein.
 22. Themethod for purifying NS1 protein as claimed in claim 27, wherein theflavivirus is a dengue virus.
 23. The method for purifying NS1 proteinas claimed in claim 28, wherein the flavivirus is dengue virusserotype
 1. 24. An immunogenic composition, comprising as the activeprinciple, an NS1 protein of a flavivirus, wherein the NS1 protein is inhexameric form, optionally associated with other proteins, and at leastone pharmaceutical vehicle.
 25. The immunogenic composition as claimedin claim 30, wherein the composition further comprises at least onemixture of NS1 proteins in hexameric form of a dengue virus serotype.26. A method for preparing an immunogenic composition capable ofinducing the production of antibodies in vivo comprising combining anNS1 protein in hexameric form, or an NS1 protein expressed from a systemfor the expression of NS1 protein in hexameric form, with at least onepharmaceutical vehicle.
 27. A method for manufacturing a medicinalproduct capable of inducing passive immunization comprising producing atleast one monoclonal anti-NS1 antibody having a high affinity for NS1protein in hexameric form, purifying the monoclonal antibody, andmodifying the monoclonal antibody by selecting for Fab fragments orhumanizing the monoclonal antibody, wherein the hexameric form isnondegraded.
 28. An immunogenic composition, comprising an activeprinciple and a pharmaceutical vehicle, wherein the active principle iseither a polynucleotide capable of expressing all or part of an NS1protein of a dengue virus of any serotype, or the active principle is anexpression system comprising at least one promoter capable ofexpressing, in a host into which it is injected, a DNA encoding an NS1protein of a dengue virus of any serotype.
 29. A method for expressing apolynucleotide encoding an NS1 protein of a dengue virus, comprisingassociating a polynucleotide of SEQ ID No. 1 with a promoter for saidpolynucleotide, and expressing the polynucleotide in a eukaryotic cell.